(a) Field of the Invention
The invention relates to a composition and method of application to improve the repair and to regenerate cartilaginous tissues and other tissues including without limitation meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors, and ulcers.
(b) Description of Prior Art
1) The Cartilage Repair Problem:
Cartilage: Structure, Function, Development, Pathology
Articular cartilage covers the ends of bones in diarthroidial joints in order to distribute the forces of locomotion to underlying bone structures while simultaneously providing nearly frictionless articulating interfaces. These properties are furnished by the extracellular matrix composed of collagen types II and other minor collagen components and a high content of the proteoglycan aggrecan. In general, the fibrillar collagenous network resists tensile and shear forces while the highly charged aggrecan resists compression and interstitial fluid flow. The low friction properties are the result of a special molecular composition of the articular surface and of the synovial fluid as well as exudation of interstitial fluid during loading onto the articular surface (Ateshian, 1997; Higaki et al., 1997; Schwartz and Hills, 1998).
Articular cartilage is formed during the development of long bones following the condensation of prechondrocytic mesenchymal cells and induction of a phenotype switch from predominantly collagen type I to collagen type II and aggrecan (Hall, 1983; Pechak et al., 1986). Bone is formed from cartilage when chondrocytes hypertrophy and switch to type X collagen expression, accompanied by blood vessel invasion, matrix calcification, the appearance of osteoblasts and bone matrix production. In the adult, a thin layer of articular cartilage remains on the ends of bones and is sustained by chondrocytes through synthesis, assembly and turnover of extracellular matrix (Kuettner, 1992). Articular cartilage disease arises when fractures occur due to physical trauma or when a more gradual erosion, as is characteristic of many forms of arthritis, exposes subchondral bone to create symptomatic joint pain (McCarty and Koopman, 1993). In addition to articular cartilage, cartilaginous tissues remain in the adult at several body sites such as the ears and nose, areas that are often subject to reconstructive surgery.
2) Cartilage Repair: The Natural Response
Articular cartilage has a limited response to injury in the adult mainly due to a lack of vascularisation and the presence of a dense proteoglycan rich extracellular matrix (Newman, 1998; Buckwalter and Mankin, 1997; Minas and Nehrer, 1997). The former inhibits the appearance of inflammatory and pluripotential repair cells, while the latter emprisons resident chondrocytes in a matrix non-conducive to migration. However, lesions that penetrate the subchondral bone create a conduit to the highly vascular bone allowing for the formation of a fibrin clot that traps cells of bone and marrow origin in the lesion leading to a granulation tissue. The deeper portions of the granulation tissue reconstitute the subchondral bone plate while the upper portion transforms into a fibrocartilagenous repair tissue. This tissue can temporarily possess the histological appearance of hyaline cartilage although not its mechanical properties (Wei et al., 1997) and is therefore unable to withstand the local mechanical environment leading to the appearance of degeneration before the end of the first year post-injury. Thus the natural response to repair in adult articular cartilage is that partial thickness lesions have no repair response (other than cartilage flow and localized chondrocyte cloning) while full-thickness lesions with bone penetration display a limited and failed response. Age, however, is an important factor since full thickness lesions in immature articular cartilage heal better than in the adult (DePalma et al., 1966; Wei et al., 1997) and superficial lacerations in fetal articular cartilage heal completely in one month without any involvement of vasculature or bone-derived cells (Namba et al., 1998).
3) Current Approaches for Assisted Cartilage Repair
Current clinical treatments for symptomatic cartilage defects involve techniques aimed at: 1) removing surface irregularities by shaving and debridement 2) penetration of subchondral bone by drilling, fracturing or abrasion to augment the natural repair response described above (i.e. the family of bone-marrow stimulation techniques) 3) joint realignment or osteotomy to use remaining cartilage for articulation 4) pharmacological modulation 5) tissue transplantation and 6) cell transplantation (Newman, 1998; Buckwalter and Mankin, 1997). Most of these methods have been shown to have some short term benefit in reducing symptoms (months to a few years), while none have been able to consistently demonstrate successful repair of articular lesions after the first few years. The bone marrow-stimulation techniques of shaving, debridement, drilling, fracturing and abrasion arthroplasty permit temporary relief from symptoms but produce a sub-functional fibrocartilagenous tissue that is eventually degraded. Pharmacological modulation supplying growth factors to defect sites can augment natural repair but to date insufficiently so (Hunziker and Rosenberg, 1996; Sellers et al., 1997). Allograft and autograft osteochondral tissue transplants containing viable chondrocytes can effect a more successful repair but suffer from severe donor limitations (Mahomed et al., 1992; Outerbridge et al., 1995).
4) Bone-Marrow Stimulation
The family of bone marrow-stimulation techniques include debridement, shaving, drilling, microfracturing and abrasion arthroplasty. They are currently used extensively in orthopaedic clinical practice for the treatment of focal lesions of articular cartilage that are full-thickness, i.e. reaching the subchondral bone, and are limited in size, typically less than 3 cm2 in area. Use of these procedures was initiated by Pridie and others (Pridie, 1959; Insall, 1967; DePalma et al., 1966) who reasoned that a blood clot could be formed in the region of an articular cartilage lesion by violating the cartilage/bone interface to induce bleeding from the bone into the cartilage defect that is avascular. This hematoma could then initiate the classical cascade of wound healing events that leads to successful healing or at least scarring in wounds of vascularized tissues (Clark, 1996). Variations of the Pridie drilling technique were proposed later including abrasion arthroplasty (Childers and Ellwood, 1979; Johnson, 1991) and microfracturing (Rodrigo et al., 1993; Steadman et al., 1997). Abrasion arthroplasty uses motorised instruments to grind away abnormally dense subchondral bone to reach a blood supply in the softer deeper bone. The microfracture technique uses a pick, or an awl, to pierce the subchondral bone plate deep enough (typically 3-4 mm), again to reach a vascular supply and create a blood clot inside the cartilage lesion. Practitioners of the microfracture technique claim to observe a higher success rate than drilling due to the lack of any heat-induced necrosis and less biomechanical destabilisation of the subchondral bone plate with numerous smaller fracture holes rather than large gaps in the plate producing by drilling (Steadman et al., 1998). Yet another related technique for treating focal lesions of articular cartilage is mosaicplasty or osteochondral autograft transplantation (OATS) where cartilage/bone cylinders are transferred from a peripheral “unused” region of a joint to the highly loaded region containing the cartilage lesion (Hangody et al., 1997).
There is no universal consensus among orthopaedists on which type of articular cartilage lesion should receive which type of treatment. There is also a lack of rigorous scientific studies that demonstrate the efficacy of these treatments for particular indications. Thus the choice of treatment for cartilage lesions is largely dependent on the training, inclinations and personal experience of the practitioner. Reasons for this lack of consensus are multifold but include the variability in the type of lesion treated and a variable if not uncontrolled success in the formation of a “good quality” blood clot. Some of the problems associated with forming a good quality blood clot with these procedures are 1) the uncontrolled nature of the bleeding coming from the bone, which never fills up the cartilage lesion entirely 2) platelet mediated clot contraction occurring within minutes of clot formation reduces clot size and could detach it from surrounding cartilage (Cohen et al., 1975) 3) dilution of the bone blood with synovial fluid or circulating arthroscopy fluid and 4) the fibrinolytic or clot dissolving activity of synovial fluid (Mankin, 1974). Some of these issues were the motivation behind some studies where a blood clot was formed ex vivo and then cut to size and packed into a meniscal defect (Arnoczky et al., 1988) or an osteochondral defect (Palette et al., 1992). Something similar to the classical wound healing cascade then ensued to aid healing of the defect. This approach did clearly provide more filling of the defect with repair tissue, however the quality of the repair tissue was generally not acceptable, being predominantly fibrous and mechanically insufficient. Some probable reasons for a less than satisfactory repair tissue with this approach are 1) continued platelet mediated clot contraction 2) the lack of viability of some blood components due to extensive ex vivo manipulation and 3) the solidification of the clot ex vivo which precludes good adhesion to all tissue surfaces surrounding the cartilage defect and limits defect filling. In summary, current clinical procedures practised by orthopaedists for treating focal lesions of articular cartilage mostly depend on the formation of a blood clot within the lesion. However the ability to form a good quality blood clot that fills the lesion and contains all of the appropriate elements for wound healing (platelets, monocytes, fibrin network etc) in a viable state produces inconsistent and often unsatisfactory outcomes. One of the embodiments of the present invention ameliorates this situation by providing a composition and method for delivering these blood borne wound healing elements in a full-volume non-contracting matrix to an articular cartilage lesion.
5) Biomaterials and Growth Factors
Several experimental techniques have been proposed to repair cartilage lesions using biomaterials and growth factors, sometimes each alone but often in combination. The analogy with the above-described family of bone-marrow stimulation techniques is clear. The fibrin scaffold of the blood clot could be replaced with a prefabricated biomaterial scaffold and the natural mitogenic and chemotactic factors in the blood clot could be replaced with user-controlled quantities and species of soluble elements such as recombinant growth factors. Examples of this approach include the use of fibrin glues to deliver recombinant proteins such as insulin-like-growth factors (Nixon et al., 1999) and transforming growth factors (Hunziker and Rosenberg, 1996). Other biologics have been combined with generic biomaterials such as polylactic acid (PLA), Polyglycolic acid (PGA), collagen matrices and fibrin glues including bone morphogenetic proteins (Sellers et al., 1997; Sellers, 2000; Zhang et al. Patent WO 00/44413, 2000), angiotensin-like peptides (Rodgers and Dizerega, Patent WO 00/02905, 2000), and extracts of bone containing a multiplicity of proteins called bone proteins or BP (Atkinson, Patent WO 00/48550, 2000). In the latter method, BP soaked collagen sponges needed to be held in the cartilage defect using an additional fibrin/thrombin based adhesive, creating a rather complex and difficult to reproduce wound healing environment. Coating the biomaterial with fibronectin or RGD peptides to aid cell adhesion and cell migration has been done (Breckke and Coutts, U.S. Pat. No. 6,005,161, 1999). Some previous methods have combined bone-marrow stimulation with post-surgical injection of growth hormone in the synovial space with limited success (Dunn and Dunn, U.S. Pat. No. 5,368,051, 1994). Specific biomaterials compositions have also been proposed such as mixtures of collagen, chitosan and glycoaminoglycans (Collombel et al., U.S. Pat. No. 5,166,187, 1992; Suh et al., Patent WO 99/47186, 1999), a crushed cartilage and bone paste (Stone, U.S. Pat. No. 6,110,209, 2000), a multicomponent collagen-based construct (Pahcence et al., U.S. Pat. No. 6,080,194, 2000) and a curable chemically reactive methacrylate-based resin (Braden et al., U.S. Pat. No. 5,468,787, 1995). None of these approaches has reached the clinic due to their inability to overcome some of the following problems 1) lack of retention and adherence of the biomaterial in the cartilage defect 2) lack of sustained release of active forms of these molecules at effective concentrations over prolonged periods of time 3) multiple and uncontrolled biological activities of the delivered molecules 4) cytotoxicity of acidic degradation products of PGA and PLA 5) inappropriate degradation kinetics or immunogencity of the carrier biomaterial and 6) undesirable systemic or ectopic affects (calcification of organs) of the active biologics. The successful implementation of these approaches awaits the solution to some or all of these issues.
6) Cell Transplantation
Techniques involving cell transplantation have provoked much recent interest due to their ability to enhance cartilage repair by introducing into articular defects, after ex vivo passaging and manipulation, large numbers of autologous chondrocytes (Grande et al., 1989; Brittberg et al., 1994 and 1996; Breinan et al., 1997), allogenic chondrocytes (Chesterman and Smith, 1968; Bently and Greer, 1971; Green, 1977; Aston and Bently, 1986; Itay et al., 1987; Wakatini et al., 1989; Robinson et al., 1990; Freed et al., 1994; Noguchi et al., 1994; Hendrickson et al., 1994; Kandel et al., 1995; Sams and Nixon, 1995; Specchia et al., 1996; Frankel et al., 1997; Hyc et al. 1997; Kawamura et al., 1998), xenogenic chondrocytes (Homminga et al., 1991), perichondrial cells (Chu et al., 1995; Chu et al., 1997), or autogenic and allogenic bone marrow-derived mesenchymal stem cells (Wakatini et al., 1994; Butnariu-Ephrat, 1996; Caplan et al., 1997; Nevo et al., 1998). The cell transplantation approach possesses some potential advantages over other cartilage repair techniques in that they 1) minimise additional cartilage and bone injury, 2) reduce reliance on donors by ex vivo cell production, 3) could mimic natural biological processes of cartilage development, and 4) may provide tailored cell types to execute better repair. One technique using autologous chondrocytes is in the public domain and is commercially available having been used in several thousand US and Swedish patients (http://www.genzyme.com). In this technique chondrocytes are isolated from a cartilage biopsy of a non-load bearing area, proliferated during several weeks, and re-introduced into the cartilage lesion by injection under a sutured and fibrin-sealed periosteal patch harvested from the patient's tibia. Knowledge of its efficacy has been questioned (Messner and Gillquist, 1996; Brittberg, 1997; Newman, 1998) and is unfortunately not known due to the lack of completion of an FDA requested controlled and randomised clinical trial. Recent animal studies indicate that the injected passaged autologous chondrocytes contribute very little to the observed healing and that the outcome is similar to that obtained using bone-marrow stimulation (Breinan et al., 1997 and Breinan et al., 2000). Thus the surgical preparation of the defect could be the main factor inducing repair, in this procedure as well. Nonetheless, due to the enormous potential benefit of cell transplantation, a large number of patents have been granted in the past two years to protect aspects of autologous chondrocyte processing (Tubo et al., U.S. Pat. No. 5,723,331, 1998; Villeneuve, U.S. Pat. No. 5,866,415, 1999), as well as the use and preparation of adipocytes (Mueller and Thaler, U.S. Pat. No. 5,837,235, 1998; Halvorsen et al., Patent EP 1 077 253, 2001), hematopoeitic precursors (Peterson and Nousek-Goebl, U.S. Pat. No. 6,200,606, 2001), amniotic membrane cells (Sackier, 1997), mesenchymal stem cells (Caplan and Haynesworth, U.S. Pat. No. 5,811,094, 1998; Naughton and Naughton, U.S. Pat. No. 5,785,964, 1998; Naughton and Willoughby, U.S. Pat. No. 5,842,477, 1998; Grande and Lucas, U.S. Pat. No. 5,906,934, 1999; Johnstone and Yoo, U.S. Pat. No. 5,908,784, 1999), and general techniques using chondrocytes/fibroblasts and their progenitors, epithelial cells, adipocytes, placental cells and umbilical cord blood cells (Purchio et al., U.S. Pat. No. 5,902,741, 1999), all for use in cartilage repair.
7) The Cell Delivery Problem
Cell transplantation for assisted cartilage repair necessarily involves a technique to deliver and retain viable and functional transplanted cells at the site of injury. When cells are grown ex vivo with or without a support matrix, press-fitting may be used by preparing an implant that is slightly larger than the defect and forcing it therein (Aston and Bentley 1986; Wakatini et al., 1989; Freed et al., 1994; Chu et al., 1997; Frankel et al., 1997; Kawamura et al., 1998). Press-fitting necessitates the use of a tissue that is formed ex vivo and thus not optimised for the geometric, physical, and biological factors of the site in which it is implanted. Suturing or tacking the implant can aid retention (Sams and Nixon, 1995) although sutures are known to be an additional injury to the articular surface inducing yet another limited repair process (Breinan et al., 1997). Biological glues have been attempted with limited success (Kandel et al., 1995; Jurgenson et al., 1997). When the implant is not amenable to press fitting, such as with contracting collagen gels or fibrin clots, or when cells alone without a support matrix are implanted, often a sutured patch of periosteum or another similar tissue is used to retain the implant material within the defect site (Grande et al., 1989; Brittberg et al., 1994; Grande et al., 1989; Brittberg et al., 1996; Breinan et al., 1997). Such a technique may benefit from an ability of the periosteum to stimulate cartilage formation (O'Driscoll et al., 1988 and 1994), but suffers again from the introduction of sutures and the complex nature of the operation involving periosteal harvesting and arthrotomy. Cells have also been delivered to deep full thickness defects using a viscous hyaluronic acid solution (Robinson et al., 1990; Butnariu-Ephrat, 1996). As with cell sources for cartilage repair, there are several recently published patents for delivery vehicles in cartilage repair ranging from gel matrices (Griffith et al., 1998; Caplan et al., 1999), to sutures and fibres (Vacanti et al., 1998; Vacanti and Langer, 1998a and 1998b), to screw type devices (Schwartz, 1998), and magnetic systems (Halpern, 1997). Taking together the above, current cell delivery techniques for cartilage repair are clearly not optimal. A desirable cell delivery vehicle would be a polymeric solution loaded with cells which solidifies when injected into the defect site, adheres and fills the defect, and provides a temporary biodegradable scaffold to permit proper cell differentiation and the synthesis and assembly of a dense, mechanically functional articular cartilage extracellular matrix.
8) Repair of Other Tissues Including Meniscus, Ligament, Tendon, bone, skin, cornea, periodontal tissues, Abscesses, Resected Tumours, and Ulcers
Natural and assisted repair of musculoskeletal and other tissues are very broad fields with numerous complex biological processes and a wide variety of approaches to accelerate the repair process (as in bone repair), aid it in tissues that have little intrinsic repair capacity (as in cartilage repair), and to reduce scarring (as in burn treatments) (Clark, 1996). Although differences certainly occur in the biological elements and processes involved, the global events in (non-fetal) wound repair are identical. These include the formation of a blood clot at the site of tissue disruption, release of chemotactic and mitogenic factors from platelets, influx of inflammatory cells and pluripotential repair cells, vascularisation, and finally the resolution of the repair process by differentiation of repair cells their synthesis of extracellular matrix components. In a successful repair outcome the specific local tissue environment and the specific local population of pluripotential repair cells will lead to the formation of the correct type of tissue, bone to replace bone, skin to replace skin etc. Given the similarity of the general elements in the tissue repair process, it is not surprising that approaches to aid repair in one tissue could also have some success in aiding repair in other tissues. This possibility becomes much more likely if the method and composition to aid repair is based upon augmenting some aspect of the natural wound healing cascade without significantly deviating from this more or less optimised sequence of events. In the present invention particular composition and methods are proposed to provide a more effective, adhesive, and non-contracting blood clot at the site of tissue repair. Examples and preferred embodiments are shown for cartilage repair, one of the most difficult tissues to repair. However application of the composition and method and modifications thereof, conserving the same basic principles, to aid repair of other tissues including meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumours, and ulcers, are obvious to those who are skilled in the art.
9) Use of Chitosan in Pharmaceuticals, Wound Healing, Tissue Repair and as a Hemostatic Agent
Chitosan, which primarily results from the alkaline deacetylation of chitin, a natural component of shrimp and crab shells, is a family of linear polysaccharides that contains 1-4 linked glucosamine (predominantly) and N-acetyl-glucosamine monomers (Austin et al., 1981). Chitosan and its amino-substituted derivatives are pH-dependent, bioerodible and biocompatible cationic polymers that have been used in the biomedical industry for wound healing and bone induction (Denuziere et al., 1998; Muzzarelli et al., 1993 and 1994), drug and gene delivery (Carreno-Gomez and Duncan, 1997; Schipper et al., 1997; Lee et al., 1998; Bernkop-Schnurch and Pasta, 1998) and in scaffolds for cell growth and cell encapsulation (Yagi et al, 1997, Eser Elcin et al., 1998; Dillon et al., 1998; Koyano et al., 1998; Sechriest et al., 2000; Lahiji et al 2000; Suh et al., 2000). Chitosan is termed a mucoadhesive polymer (Bernkop-Schnurch and Krajicek, 1998) since it adheres to the mucus layer of the gastrointestinal epithelia via ionic and hydrophobic interactions, thereby facilitating peroral drug delivery. Biodegradability of chitosan occurs via its susceptibility to enzymatic cleavage by chitinases (Fukamizo and Brzezinski, 1997), lysozymes (Sashiwa et al., 1990), cellulases (Yalpani and Pantaleone, 1994), proteases (Terbojevich et al., 1996), and lipases (Muzzarelli et al., 1995). Recently, chondrocytes have been shown to be capable of expressing chitotriosidase (Vasios et al., 1999), the human analogue of chitosanase; its physiological role may be in the degradation of hyaluronan, a linear polysaccharide possessing some similarity with chitosan since it is composed of disaccharides of N-acetyl-glucosamine and glucuronic acid.
Chitosan has been proposed in various formulations, alone and with other components, to stimulate repair of dermal, corneal and hard tissues in a number of reports (Sall et al., 1987; Bartone and Adickes, 1988; Okamoto et al., 1995; Inui et al., 1995; Shigemasa and Minami, 1996; Ueno et al., 1999; Cho et al., 1999; Stone et al., 2000; Lee et al., 2000) and inventions (Sparkes and Murray, U.S. Pat. No. 4,572,906, 1986; Mosbey, U.S. Pat. No. 4,956,350, 1990; Hansson et al., U.S. Pat. No. 5,894,070, 1999; Gouda and Larm, U.S. Pat. No. 5,902,798, 1999; Drohan et al., U.S. Pat. No. 6,124,273, 2000; Jorgensen WO 98/22114, 1998). The properties of chitosan that are most commonly cited as beneficial for the wound repair process are its biodegradability, adhesiveness, prevention of dehydration and as a barrier to bacterial invasion. Other properties that have also been claimed are its cell activating and chemotractant nature (Peluso et al., 1994; Shigemasa and Minami, 1996; Inui et al., 1995) its hemostatic activity (Malette et al., 1983; Malette and Quigley, U.S. Pat. No. 4,532,134, 1985) and an apparent ability to limit fibroplasia and scarring by promoting a looser type of granulation tissue (Bartone and Adickes, 1988; Stone et al., 2000). Although a general consensus about the beneficial effects of chitosan in wound healing is apparent, its exact mechanism of action is not known, nor is the most effective means of its application, i.e. as a powder, suspension, sponge, membrane, solid gel etc. Part of the reason for the ambiguity in its mechanism of action could be that many previous studies used chitosan that was not chemically defined (acetyl content and distribution, molecular weight) and of unknown purity. The interesting hemostatic potential of chitosan has also led to its direct application to reduce bleeding at grafts and wound sites (Malette et al., 1983; Malette and Quigley, U.S. Pat. No. 4,532,134, 1985). Some studies claim that the hemostatic activity of chitosan derives solely from it's ability to agglutinate red blood cells (Rao and Sharma, 1997) while others believe its polycationic amine character can activate platelets to release thrombin and initiate the classical coagulation cascade thus leading to its use as a hemostatic in combination with fibrinogen and purified autologous platelets (Cochrum et al. U.S. Pat. No. 5,773,033, 1998). In the context of the present invention, it is important to note in these reports and inventions a complete lack of any example where blood was mixed with chitosan in solution and applied therapeutically to aid tissue repair through the formation of a chitosan containing blot clot at the repair site.
One technical difficulty that chitosan often presents is a low solubility at physiological pH and ionic strength, thereby limiting its use in a solution state. Thus typically, dissolution of chitosan is achieved via the protonation of amine groups in acidic aqueous solutions having a pH ranging from 3.0 to 5.6. Such chitosan solutions remain soluble up to a pH near 6.2 where neutralisation of the amine groups reduces interchain electrostatic repulsion and allows attractive forces of hydrogen bonding, hydrophobic and van der Waals interactions to cause polymer precipitation at a pH near 6.3 to 6.4. A prior invention (Chenite Patent WO 99/07416; Chenite et al., 2000) has taught that admixing a polyol-phosphate dibasic salt (i.e. glycerol-phosphate) to an aqueous solution of chitosan can increase the pH of the solution while avoiding precipitation. In the presence of these particular salts, chitosan solutions of substantial concentration (0.5-3%) and high molecular weight (<several hundred kDa) remain liquid, at low or room temperature, for a long period of time with a pH in a physiologically acceptable neutral region between 6.8 and 7.2. This aspect facilitates the mixing of chitosan with cells in a manner that maintains their viability. An additional important property is that such chitosan/polyol-phosphate (C/PP) aqueous solutions solidify or gel when heated to an appropriate temperature that allows the mixed chitosan/cell solutions to be injected into body sites where, for example cartilage nodules can be formed in subcutaneous spaces in nude mice (Chenite et al., 2000). It is important to note that some other studies have retained chitosan in a soluble state at physiological pH but these studies necessitated the reduction of either chitosan concentration (to 0.1% in Lu et al Biomaterials 1999) or of chitosan molecular weight and degree of deacetylation (to −350 kD and 50% in respectively in Cho et al Biomaterials, 1999) Other studies have also shown that chitosan presents a microenvironment that supports the chondrocyte and osteoblast phenotype (Sub et al., 2000; Lahiji at al., 2000; Seichrist et al., 2000) however these studies were not based on liquid chitosan in a form that could be mixed with cells and injected. Finally NN-dicarboxylmethyl chitosan sponges have been soaked with BMP7 and placed into osteochondral defects of rabbits (Mattioli-Belmonte, 1999). Here again some improved histochemical and immunohistochemical outcome was observed, however, incomplete filling of the defect with repair tissue and a significant difficulty in retaining the construct within the defect appeared to be insurmountable problems. The present invention overcomes these issues and presents several novel solutions for the delivery of compositions for the repair of cartilage and other tissues.
10) Summary of Prior Art
In summary of prior art for assisted cartilage repair, it may be said that many techniques to improve the very limited natural repair response of articular cartilage have been proposed and experimentally tested. Some of these techniques have achieved a certain level of acceptance in clinical practice but this has mainly been so due to the absence of any practical and clearly effective method of improving the repair response compared to that found when the family of bone marrow stimulation techniques is applied. This invention addresses and solves several of the main problematic issues in the use of cells and blood components to repair articular cartilage. One main obstacle towards the development of an effective cartilage repair procedure is the absence of a composition and method to provide an appropriate macromolecular environment within the space requiring cartilage growth (cartilage defect or other site requiring tissue bulking or reconstruction). This macromolecular environment or matrix should 1) be amenable to loading with active biological elements (cells, proteins, genes, blood, blood components) in a liquid state 2) then be injectable into the defect site to fill the entire defect or region requiring cartilage growth 3) present a primarily nonproteinaceous environment to limit cell adhesion and cell-mediated contraction of the matrix, both of which induce a fibrocytic cellular phenotype (fibrous tissue producing) rather than chondrocytic cellular phenotype (cartilaginous tissue producing) and which can also disengage the matrix from the walls of the defect 4) be cytocompatible, possessing physiological levels of pH and osmotic pressure and an absence of any cytotoxic elements 5) be degradable but present for a sufficiently long time to allow included biologically active elements to fully reconstitute a cartilaginous tissue capable of supporting mechanical load without degradation. In addition it is obvious to those skilled in the art that such a combination of characteristics could be applied with minimal modifications towards the repair of other tissues such as meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors, and ulcers.
It would be highly desirable to be provided with a new composition for use in repair and regeneration of cartilaginous tissues.